Otoconia, crystallites of CaCO3 and proteins, are required for an individual to sense linear acceleration and gravity for spatial orientation and balance control. Dysfunction of this system is a critical clinical problem, particulady in the elderly, yet little is known about the mechanisms that regulate otoconial biosynthesis. Otoconia appear to be in a dynamic state of equilibrium highly susceptible to adverse effects of aging, infections, diseases and genetic mutations, but the molecular mechanisms of these problems are largely unknown. Our overall hypotheses are that (1) the interplay of multiple specific protein components of otoconia and their embedding membrane determines their site-specific formation; (2) Otoconial protein homeostasis is essential to normal otoconia function; and (3) OC90, a major protein component of otoconia, is essential for the formation of otoconia by sequestering Ca. OC90 mutant mice will manifest a functional deficit of the inner ear, with the vestibule as the focus of this proposal. Specifically in this proposal, we will (1) identify other murine otoconial proteins and their cellular origin, to help identify factors that may contribute to otoconia formation. We will identify any interactions among OC90, otogelin, and other proteins in otoconia, its embedding membrane or the sensory epithelium by co-precipitation on PS10 protein chips to test hypothesis #1. (2) We will examine whether disrupted otoconin homeostasis caused the abnormal and malfunctioning otoconia in aging (C57BI/6J) and degenerating otoconia (head-tilt mice) by protein profiling on H4 chips, and examine changes in the deposition of otoconial proteins in embryos and newborn pups that have disrupted calcium homeostasis (tilt, PMCA-/- mice). (3) Then we will test hypothesis #3 by examining the spatial coordination of OC90 expression and Ca transportation, and by making two mutant mouse lines, one null and the other carting only one functional (PLA2L) domain, to study the role of OC90 in otoconia formation and gravity sensing, as well as in cochlea function. Combined with future sophisticated behavioral, physiological and gene/protein profiling studies, we will demonstrate the role of OC90 in bodily balancing, the molecular mechanisms of site-specific formation, regulation and proper maintenance of otoconia. Thus we will contribute to understanding inner ear development, function, physiology and pathology.